These studies are intended primarily to characterize the mechanisms involved in active calcium transport by renal epithelia. Proposed experiments include examination of the membrane processes and second messengers involved in parathyroid hormone (PTH) stimulation of calcium absorption in renal cortical thick ascending limbs of Henle (cTAL), a nephron site of the physiological hypercalcemic action of PTH. In order to accomplish this characterization, electrophysiological techniques combined with digital fluorescence imaging techniques will be applied to single perfused segments of renal tubules. Biochemical and isotopic flux studies will be performed on cultured Madin-Darby canine kidney (MDCK) cells. Fluorescence ratio microscopy imaging will be used to measure cytosolic calcium in single perfused thick limbs. This technology is established in our lab and preliminary feasibility data are included. Other pilot studies described in this application support the use of confluent MDCK cells as a model with which to examine PTH-sensitive calcium transport. We have established that PTH, but not calcitonin, specifically stimulates transepithelial calcium transport in MDCK monolayers grown to confluence on porous filters. Further, challenge with PTH triggers phosphatidyl inositol hydrolysis in these cells. Thus, from a physiological standpoint MDCK cells simulate completely the response to PTH in cortical thick limbs. However, unlike cTALs, MDCK cells can be grown in sufficient number to permit detailed biochemical analysis and characterization of the receptor events involved in mediating the response to PTH. Studies have been designed to test hypotheses related to: 1) regulation of cytosolic calcium in PTH-sensitive, calcium transporting cTALs; 2) PTH-induced second messengers; and, 3) regulation of PTH-dependent calcium transport by prostaglandins, and other factors known to alter distal calcium transport in vivo, including the concentration of calcium in and ionic composition of extracellular fluid. These studies should provide valuable information on the regulation of intracellular calcium in PTH-sensitive distal target cells, characterization of receptors and second messengers involved in active calcium absorption, and insight into down-regulation and other regulatory factors modulating physiological PTH action in target renal cortical thick limbs and in a cell culture model of distal tubule cells.